With respect to a multienzyme complex of deoxyribonucleoside triphosphate
(dNTP) synthesis somehow juxtaposed with DNA replication sites, our laboratory
has demonstrated the existence of a multienzyme complex in T4-infected E. coli,
named the T4 dNTP synthetase complex, but the idea of direct linkage of dNTP
synthesis to DNA replication and organization of the complex has not been well
established. This study had two objectives. The first objective was to test the specific
hypothesis that gp32, the single-stranded DNA binding protein encoded by gene 32,
plays a role in recruiting enzymes of dNTP synthesis to the replisome and in
organizing the dNTP synthetase complex. By use of two newly created gene 32
mutants along with several experimental approaches, DNA-cellulose
chromatography, coimmunoprecipitation, and glutathione-S-transferase pull downs,
interactions of gp32 with thymidylate synthase (gptd), ribonucleotide reductase
(gpnrdA/B), and E. coli NDP kinase have been identified. These results support the
hypothesis that gp32 helps to recruit enzymes of dNTP synthesis to DNA replication
sites.
As the second objective, I investigated contributions of two host proteins, E. coli
nueleoside diphosphate kinase (NDP kinase) and adenylate kinase (Adk), to the
organization of the complex. As an important step to understand roles of E. coli NDP
kinase in the complex, I identified direct interactions of E. coli NDP kinase with
gpnrdA/B, dCMP hydroxymethylase (gp42), and dihydrofolate reductase (gpfrd) by
means of coimmunoprecipitation and glutathione-S-transferase pull-down
experiments. Interestingly, these interactions were influenced by the presence of
substrate nucleotides or an analog for E. coli NDP kinase, suggesting that metabolite
flux may affect the preference of E. coli NDP kinase binding to enzymes in the
complex in vivo. Meanwhile, Adk involvement in DNA precursor synthesis has been
suggested, particularly in phage T4-infected E. coli, from observations of increased
thermostability of temperature-sensitive Adk in situ. The involvement of E. coil Adk
in the complex was demonstrated by identifying some proteins of the T4 dNTP
synthetase complexgp42, dNMP kinase (gpl), gpfrd, and E. coli NDP
kinasedirectly interacting with Adk, implying that E. coil Adk would be properly
located in the complex to efficiently carry out the conversion of dNDPs to dNTPs.
This implication was supported by measurements of T4 DNA synthesis.
Taken together, this research strongly supports the idea of connection of dNTP
synthesis to DNA replication and allows us to take a step toward understanding the
organization of the complex at DNA replication sites. / Graduation date: 2005
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/29969 |
| Date | 02 February 2005 |
| Creators | Kim, JuHyun |
| Contributors | Mathews, Christopher K. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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